%0 Journal Article
%A Behnke, Jörg
%A Eskelinen, Eeva-Liisa
%A Saftig, Paul
%A Schröder, Bernd
%T Two dileucine motifs mediate late endosomal/lysosomal targeting of transmembrane protein 192 (TMEM192) and a C-terminal cysteine residue is responsible for disulfide bond formation in TMEM192 homodimers
%D 2011
%R 10.1042/BJ20101396
%J Biochemical Journal
%P 219-231
%V 434
%N 2
%X TMEM192 (transmembrane protein 192) is a novel constituent of late endosomal/lysosomal membranes with four potential transmembrane segments and an unknown function that was initially discovered by organellar proteomics. Subsequently, localization in late endosomes/lysosomes has been confirmed for overexpressed and endogenous TMEM192, and homodimers of TMEM192 linked by disulfide bonds have been reported. In the present study the molecular determinants of TMEM192 mediating its transport to late endosomes/lysosomes were analysed by using CD4 chimaeric constructs and mutagenesis of potential targeting motifs in TMEM192. Two directly adjacent N-terminally located dileucine motifs of the DXXLL-type were found to be critical for transport of TMEM192 to late endosomes/lysosomes. Whereas disruption of both dileucine motifs resulted in mistargeting of TMEM192 to the plasma membrane, each of the two motifs was sufficient to ensure correct targeting of TMEM192. In order to study disulfide bond formation, mutagenesis of cysteine residues was performed. Mutation of Cys266 abolished disulfide bridge formation between TMEM192 molecules, indicating that TMEM192 dimers are linked by a disulfide bridge between their C-terminal tails. According to the predicted topology, Cys266 would be localized in the reductive milieu of the cytosol where disulfide bridges are generally uncommon. Using immunogold labelling and proteinase protection assays, the localization of the N- and C-termini of TMEM192 on the cytosolic side of the late endosomal/lysosomal membrane was experimentally confirmed. These findings may imply close proximity of the C-termini in TMEM192 dimers and a possible involvement of this part of the protein in dimer assembly. Abbreviations: B/T, ratio, Bound/Total ratio; CD4–Cterm, chimaera of CD4 and the C-terminus of TMEM192; CD4–Nterm, chimaera of CD4 and the N-terminus of TMEM192; CD4–Nterminv, chimaera of CD4 and the inverted N-terminus of TMEM192; CD-M6PR, cation-dependent mannose 6-phosphate receptor; CI-M6PR, cation-independent mannose 6-phosphate receptor; CKII, casein kinase II; DTT, dithiothreitol; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GGA, protein, Golgi-localized, γ-ear-containing, Arf-binding protein; HA, haemagglutinin; immuno-EM, immuno-electron microscopy; LAMP, lysosomal-associated membrane protein; LRP, LDL (low-density lipoprotein)-receptor-related protein; PBS-CM, PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2; sulfo-NHS-SS-biotin, sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; TGN, trans-Golgi network; TMEM192, transmembrane protein 192
%U http://www.biochemj.org/content/ppbiochemj/434/2/219.full.pdf